CN105229137A - Microorganism detection sensor, its manufacture method and polymer layer - Google Patents
Microorganism detection sensor, its manufacture method and polymer layer Download PDFInfo
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- CN105229137A CN105229137A CN201480018305.3A CN201480018305A CN105229137A CN 105229137 A CN105229137 A CN 105229137A CN 201480018305 A CN201480018305 A CN 201480018305A CN 105229137 A CN105229137 A CN 105229137A
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- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
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- G01N33/54373—Apparatus specially adapted for solid-phase testing involving physiochemical end-point determination, e.g. wave-guides, FETS, gratings
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- C—CHEMISTRY; METALLURGY
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
- C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
- C12M41/30—Means for regulation, monitoring, measurement or control, e.g. flow regulation of concentration
- C12M41/36—Means for regulation, monitoring, measurement or control, e.g. flow regulation of concentration of biomass, e.g. colony counters or by turbidity measurements
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- C—CHEMISTRY; METALLURGY
- C25—ELECTROLYTIC OR ELECTROPHORETIC PROCESSES; APPARATUS THEREFOR
- C25D—PROCESSES FOR THE ELECTROLYTIC OR ELECTROPHORETIC PRODUCTION OF COATINGS; ELECTROFORMING; APPARATUS THEREFOR
- C25D5/00—Electroplating characterised by the process; Pretreatment or after-treatment of workpieces
- C25D5/34—Pretreatment of metallic surfaces to be electroplated
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- C—CHEMISTRY; METALLURGY
- C25—ELECTROLYTIC OR ELECTROPHORETIC PROCESSES; APPARATUS THEREFOR
- C25D—PROCESSES FOR THE ELECTROLYTIC OR ELECTROPHORETIC PRODUCTION OF COATINGS; ELECTROFORMING; APPARATUS THEREFOR
- C25D5/00—Electroplating characterised by the process; Pretreatment or after-treatment of workpieces
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- G—PHYSICS
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- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
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- G—PHYSICS
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- G01N2600/00—Assays involving molecular imprinted polymers/polymers created around a molecular template
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Abstract
The invention provides the sensor detecting microorganism, described sensor is for test section (17), described test section (17) comprises detection electrode (11) and is arranged on the polymer layer (14) on described detection electrode, and described polymer layer (14) has the template (15) with the three-dimensional structure of the three-dimensional arrangement complementation of the microorganism of detected object (13); The trap state that described sensor is captured to described template (15) based on microorganism detects microorganism.Described polymer layer (14) is that the manufacture method by comprising following operation is formed: polymerization process (St1), under the existence of the microorganism (13) as detected object, by monomer polymerization, detection electrode forms the polymer layer (14) of catching the state of microorganism (13); And destroy operation (St2), the solution of the enzyme of contact at least partially containing bacteriolyze of the microorganism (13) being captured to polymer layer (14) is destroyed.
Description
Technical field
The present invention relates to microorganism detection sensor, its manufacture method and polymer layer.
Background technology
In recent years, in hospitality industry, grocery trade, agricultural, livestock products, cultivation, water processing establishment etc., all microorganism detection is shown great attention to.The contaminating microorganisms existed in food, pharmaceuticals, agricultural chemicals etc., even if exist with trace, also can make a big impact to the health of people.In addition, the microbial contamination in hospital, geriatric care facility becomes social concern.Further, as can be seen from the multiple circulation of antibacterial commodity, the surging of demand, in general family, also hygiene control is shown great attention to.Such as, in the occasion of food processing plant, the food dispatched from the factory is implemented to sampling bacterioscopy, implemented bacterioscopy etc. to the environment in factory, but when being measured by culture method, need cost 24-48 hours just can obtain result, become the major reason that the keeping cost to before dispatching from the factory uprises, so need detection method rapidly.Further, be also like this at agriculture field, such as, if the bacterial count in the nutrient solution of water planting increases, the risk of morbidity improves.The measures such as rapid sterilization can be taked, so detection method is effective rapidly by grasping bacterial count fast.
Due to such situation, in recent years, the necessity that can detect the technology of microbial contamination is simply surging rapidly.In addition, on-the-spot in medical treatment, the infective pathogen bacterium of transmissible disease must be identified rapidly, therefore need the technology that can detect rapidly and in high sensitivity pathogenic bacteria.As the detection of microorganism and the method for qualification, such as, there is the method such as ELISA method, western blot method.These methods such as have: after protein intrinsic to antibody (Primary antibodies) and microorganism is carried out antigen-antibody reaction, further the secondary antibody of mark and antibody (Primary antibodies) are reacted, carried out the method detected by the monitoring chemoluminescence of secondary antibody or the hydrolysis reaction of ATP.
In addition, recording in No. 2009-58232, Japanese Laid-Open Patent Publication (patent documentation 1) about utilizing the electrochemical properties with the polymkeric substance of molecular template, detecting the method coming from the Anion molecule (ATP, amino acid etc.) of microorganism.
Prior art document
Patent documentation
Patent documentation 1: No. 2009-58232, Japanese Laid-Open Patent Publication
Summary of the invention
The technical problem that invention will solve
But aforesaid method is not the method detecting microorganism itself.In addition, ELISA method etc. need to make antibody for the intrinsic protein of microorganism etc., because of but very difficult.
The object of the present invention is to provide can easy, the novel microorganism detecting sensor, its manufacture method and the polymer layer that detect microorganism in high sensitivity rapidly.
The means of technical solution problem
The invention provides the sensor detecting microorganism, described sensor is for test section, described test section comprises detection electrode and is arranged on the polymer layer on detection electrode, and described polymer layer has the template with the three-dimensional structure of the three-dimensional arrangement complementation of the microorganism of detected object; The trap state being captured to this template based on microorganism detects microorganism.Above-mentioned polymer layer is formed by following manufacture method: polymerization process, under the existence of the microorganism as detected object, by monomer polymerization, detection electrode forms the polymer layer of catching the state of microorganism; And, destroy operation, the solution of the enzyme of contact at least partially containing bacteriolyze of the microorganism being captured to polymer layer is destroyed.The preferred implementation of the sensor is: destroy the solution used in operation and contain sequestrant further.
The preferred implementation of the sensor is: described sensor also comprises quartz-crystal resonator (crystal shake Move), described quartz-crystal resonator using the detection electrode of test section as in electrode, by the change of the resonant frequency of quartz-crystal resonator, the quality change measuring polymer layer detects the trap state of microorganism.
In the sensor, above-mentioned monomer is preferably selected from the group in being made up of pyrroles, aniline, thiophene and their derivative, is preferably further made up of pyrroles's or derivatives thereof.
In the sensor, the forming surface of the above-mentioned polymer layer of above-mentioned detection electrode is preferably asperities.
In the sensor, as mentioned microorganism, preferably overall or surperficial electric charge is the microorganism of the superfluous state of negative charge.
In addition, the invention provides the manufacture method of the sensor detecting microorganism, described sensor is for test section, described test section comprises detection electrode and is arranged on the polymer layer on detection electrode, described polymer layer has the template with the three-dimensional structure of the three-dimensional arrangement complementation of microorganism, described manufacture method comprises following operation: polymerization process, under the existence of the microorganism as detected object, by monomer polymerization, detection electrode forms the polymer layer of catching the state of microorganism; And destruction operation, the solution of the enzyme of contact at least partially containing bacteriolyze of the microorganism being captured to polymer layer is destroyed.
In addition, the invention provides the polymer layer had with the template of the three-dimensional structure of the three-dimensional arrangement complementation of microorganism, described polymer layer is by comprising the manufacture method manufacture of following operation: polymerization process, under the existence of microorganism, by monomer polymerization, form polymer layer; And destruction operation, the solution of the enzyme of microorganism contact containing bacteriolyze being captured to polymer layer is destroyed.
Invention effect
Sensor of the present invention can rapidly easy, detect microorganism in high sensitivity.In addition, provide can easy, the sensor that detects microorganism in high sensitivity rapidly for the manufacture method of sensor of the present invention.
Accompanying drawing explanation
Fig. 1 schematically shows the figure of the preferred fabrication operation of the polymer layer of sensor of the present invention, Fig. 1 (a) is the sectional view before polymerization process, Fig. 1 (b) is the sectional view after polymerization process, and Fig. 1 (c) is the sectional view after destroying operation.
Fig. 2 briefly expresses in sensor of the present invention, target microorganism is caught in the schematic diagram of the appearance of template, Fig. 2 (a) is for indicating figure when target microorganism, and Fig. 2 (b) represents figure when not having target microorganism.
Fig. 3 represents the schematic diagram of the brief configuration of qcm sensor of the present invention.
Fig. 4 is the electron micrograph of Pseudomonas aeruginosa.
Fig. 5 is the electron micrograph on the polypyrrole layer surface in embodiment 1 after polymerization process.
Fig. 6 is after the destruction operation of embodiment 1, with the electron micrograph on the polypyrrole layer surface after sterile water wash.
Fig. 7 is after the destruction operation of embodiment 2, with the electron micrograph on the polypyrrole layer surface after sterile water wash.
Fig. 8 represents when using the sensor of embodiment 1 to detect microorganism, the figure of the variation of resonant frequency of quartz-crystal resonator.
Fig. 9 represents when using the sensor of embodiment 2 to detect microorganism, the figure of the variation of resonant frequency of quartz-crystal resonator.
Embodiment
Sensor of the present invention is the sensor detecting microorganism, described sensor is for test section, described test section comprises detection electrode and is arranged on the polymer layer on detection electrode, and described polymer layer has the template with the three-dimensional structure of the three-dimensional arrangement complementation of microorganism; The trap state being captured to this template based on microorganism detects microorganism.
The polymer layer of sensor of the present invention is formed: polymerization process by the manufacture method comprising following operation, under the existence of the microorganism (hereinafter referred to as " target microorganism ") as detected object, by monomer polymerization, detection electrode forms the polymer layer of catching the state of microorganism; And destruction operation, the solution of the enzyme of contact at least partially containing bacteriolyze of the microorganism being captured to polymer layer is destroyed.
Referring to accompanying drawing, the preferred embodiment of the present invention is described.
The making of polymer layer [in the sensor]
Fig. 1 schematically shows the sectional view of the polymer layer preferred fabrication operation of sensor of the present invention.Fig. 1 represents that use pyrroles is as embodiment during monomer.First, as shown in Fig. 1 (a), under the environment of contact detection with electrode 11, prepare the solution 12 containing microorganism 13 and pyrroles.The concentration of the monomer of the formation polymer layer contained in solution 12 can be 1mM-100M, and the concentration of microorganism 13 can be 1-1 × 10
10cfu/ml.
In polymerization process (St1), using detection with electrode 11 as anode, as negative electrode, electrolysis is carried out to electrode (not shown), by carrying out the oxidative polymerization of pyrroles, detection electrode 11 forms the polymer layer 14 that polypyrrole (in Fig. 1 (b), " PPy " is the abbreviation of polypyrrole) is formed.Microorganism 13 is caught in the polymer layer 14 formed.Can think, because in polymerization process, pyrroles discharges electronics on detection electrode 11, and thus pyrroles itself is with positive charge, and in order to compensate this positive charge, overall or surperficial electric charge is that the microorganism 13 of the superfluous state of negative charge is caught in polymer layer 14.
Then, as shown in Fig. 1 (c), in destruction operation (St2), the destruction operation that the microorganism 13 carrying out polymer layer 14 to catch is destroyed.Destruction operation can be carried out by making the solution of the enzyme containing bacteriolyzes such as N,O-Diacetylmuramidases contact microorganism 13.By described destruction operation, microorganism 13 is destroyed, and microorganism 13 discharges from polymer layer 14.Destroying in the solution used in operation, preferably further containing sequestrant.As destroying the sequestrant used in operation, can enumerate: EDTA (ethylenediamine tetraacetic acid (EDTA)), EGTA, NTA, DTPA, HEDTA etc.In addition, as at the enzyme destroying the bacteriolyze used in operation, can enumerate: N,O-Diacetylmuramidase, N-acetyl muramidase, achromobacter endopeptidase etc.
By being used for destroying operation by the solution of the enzyme containing bacteriolyze, the microorganism 13 that polymer layer 14 can be caught is destroyed, and destroyed microorganism 13 discharges from polymer layer 14, forms template 15.Can think, the sequestrant contained in solution makes the bacteriolysis brought by the enzyme of bacteriolyze more easily manifest, therefore, can be interpreted as: due to containing sequestrant, the destructiveness of microorganism 13 can be increased, while destroyed, easily microorganism 13 is discharged from polymer layer 14.By using the enzyme of sequestrant and bacteriolyze, easily can improve the efficiency destroying operation, as mentioned below, compared with not using the situation of sequestrant, the detection sensitivity of sensor can be promoted.The preferred 1-1000mg/ml of concentration of the enzyme of the bacteriolyze in the solution used in destruction operation.When adding sequestrant, the concentration preferred 10-1000 μ g/ml of the sequestrant in solution.In bacteriolyze operation, the solution of the enzyme containing bacteriolyze is preferably 12-48 hour with the duration of contact of the microorganism 13 being captured to polymer layer 14.In addition, in destruction operation, while above-mentioned solution contact with microorganism and carries out processing, also heat treated, ultrasonication etc. can be combined and carry out.
The region that once there is microorganism 13 in polymer layer 14 becomes the template 15 had with the three-dimensional structure of the three-dimensional arrangement complementation of microorganism 13.The polymer layer 14 possessing template 15 of such formation forms the test section 17 in sensor of the present invention with the duplexer of detection electrode 11.The thickness of the polymer layer 14 in test section 17 can be such as 0.1-10 μm.
As the microorganism 13 of detected object, as long as entirety or surperficial electric charge present the microorganism of the superfluous state of negative charge, do not limit especially.With colibacillary Escherichia, the Rhodopseudomonas of Pseudomonas aeruginosa etc., headed by the acinetobacter of Acinetobacter calcoaceticus etc., such as have: serratia, klebsiella spp, enterobacter, Chinese holly edge acidfast bacilli belongs to, Burkholderia, Sphingomonas (Sphingomonadase genus), chromobacterium, salmonella, Vibrio, legionella, Campylobacter, yersinia's genus, proteus, neisseria, Staphylococcus, streptococcus, enterococcus spp, fusobacterium, corynebacterium, listeria, bacillus, Mycobacterium, chlamydiaceae, rickettsiae, the bacterium of hemophilus.In addition, as virus, such as, have: hepatitis A virus, adenovirus, rotavirus, norovirus.As fungi, such as, have: candidiasis.As protozoon, such as, have: Cryptosporidium.The entirety of microorganism or the electric charge on surface change according to the water quality difference of the solution such as pH 12.Such as, there are the various functional groups such as carboxyl, amino, phosphate on the surface of microorganism, if pH value becomes large, then comprises the surface band negative electricity of those functional groups.Therefore, when forming template and when measuring etc., in order to become the state of negative charge surplus, such as, solution 12 is made to become alkalescence etc.
In FIG, to using pyrroles as monomer and forming polypyrrole layer and be illustrated as the situation of polymer layer, but as becoming the monomer of raw material of polymer layer, be not limited to pyrroles, other monomers such as have aniline, thiophene and their derivative etc.
The material of detection electrode 11 is not limited especially, such as, has: the multi-layered electrode, lead electrode, platinum electrode, carbon dioxide process carbon electrode etc. of the multi-layered electrode of the multi-layered electrode of the multi-layered electrode of gold electrode, Jin Hege, gold and titanium, silver electrode, silver and chromium, silver and titanium.Preferably roughened process is implemented to the face of the polymer layer 14 forming detection electrode 11.The face forming the polymer layer 14 of detection electrode 11 is asperities, makes to improve with the sticking power of polymer layer 14, in addition, also has the effect expanding electrode surface area.Such as, when use gold electrode as detection with electrode 11 when, following roughened operation can be carried out: by gold electrode surfaces implement plasma etching, thereafter by fix gold nano grain carry out roughened process.Represent with center line average roughness, the surfaceness of detection electrode surface 11 can be such as 0.4-50 μm.
[target microorganism is caught to template]
Fig. 2 briefly expresses the schematic diagram that target microorganism is caught in the appearance of template.The microorganism 13a that Fig. 2 (a) represents in sample solution is the situation of target microorganism, and the microorganism 13b that Fig. 2 (b) represents in sample solution is not the situation of target microorganism.As shown in Fig. 2 (a), Fig. 2 (b), first, under the environment contacting the test section 17 be made up of polymer layer 14 and detection electrode 11, sample solution is prepared.When electronegative microorganism is moved to the direction of test section 17 by the electrostatic interaction of the PPy film with positively charged etc., be captured in template 15 (Fig. 2 (a)) with the microorganism 13a of the three-dimensional arrangement of the three-dimensional structure complementation of template 15, but the microorganism 13b not complementary with template 15 is not captured in template 15 (Fig. 2 (b)).In addition, the movement of microorganism can be that the active of microorganism is moved, and also can be make it mobile by electrophoresis, dielectrophoresis and current etc., or make it merely precipitation or diffusion etc.In addition, even if when containing the suspended substance such as such as mud, iron rust etc. beyond microorganism in water, those suspended substances are neither identical not complementary with the 3D shape, electriferous state etc. of template 15 yet yet, thus can not be captured.Therefore, it is possible to identify target microorganism and other suspended substances.
[detection of target microorganism]
If microorganism 13a is captured in template 15, then on the duplexer be made up of polymer layer 14 and detection electrode 11, there are such as quality change, conductive characteristic change, capacitance variations, luminous reflectance factor change, temperature variation etc.In sensor of the present invention, carry out the trap state of detection template 15 pairs of microorganisms by detecting such change.So detecting target microorganism based on trap state becomes possibility.By such detection, can the rapid and highly sensitive detection of realize target microorganism.As the concrete example of the detection method of quality change, the detection method of the change of the resonant frequency detecting quartz-crystal resonator can be enumerated.Below, preferred example QCM (Quartz Crystal Microbalance) (QCM) sensor of sensor of the present invention is described.
(qcm sensor)
Fig. 3 represents the schematic diagram of the brief configuration of qcm sensor.Qcm sensor 33 possesses: the room 27 of storage solution; Be arranged on the quartz-crystal resonator 32 of the bottom of room 27; Oscillatory circuit 22; And there is the controller 21 of frequency counter.The test section 17 that quartz-crystal resonator 32 makes according to operation as shown in Figure 1, quartz wafer 24 and the order to electrode (second pair of electrode) 23 are laminated.Qcm sensor 33 possess further be immersed in sample solution 31 to electrode (pair of electrodes) 16 and reference electrode 30, test section 17 detection with electrode 11 and to electrode 16 between can connect direct supply.
First, in room 27, sample solution 31 is added.Then, apply voltage of alternating current by oscillatory circuit 22 to detection electrode 11 with between electrode 23, quartz wafer 24 is vibrated.When in the template 15 that microorganism is captured to polymer layer 14, the quality of test section 17 changes, and the resonant frequency of quartz wafer 24 changes.Frequency counter in controller 21 receives the signal of self-oscillating circuit 22, measures resonance frequency value.The trap state of microorganism is detected by the change of resonance frequency value.
Use the qcm sensor 33 shown in Fig. 3, carry out the operation shown in roughened process and Fig. 1 according to detection electrode 11 surface, polymer layer can be formed on detection electrode 11.In this case, will detection electrode 11, quartz wafer 24 be stacked gradually, the quartz-crystal resonator of electrode 23 is arranged on to the bottom of room 27, detection with electrode 11 and to electrode 16 between connect direct supply.In the formation of polymer layer using qcm sensor 33, by the variation of resonant frequency of monitoring quartz-crystal resonator while polymer layer formation, the development situation of the formation of polymer layer can be confirmed.When the microorganism of detected object exists multiple types, by forming each template of the present invention respectively and they carried out combine or by forming the template corresponding with multiple-microorganism in single template simultaneously, multiple-microorganism can be detected simultaneously.
Use sensor of the present invention, such as can in several minutes to several tens minutes bacterial detection, compared with culture method, can extremely promptly detect.In addition, owing to not using the necessary staining reagent of fluorescent dye, not using and just can detect with ATP mensuration bacterial count necessary ATP extraction reagent etc., therefore, the equipment such as water purifier, water dispenser or automatic ice maker easily assembled, easily realize automatization etc.In addition, as the bacterioscopy instrument in water examination, food inspection, may be used for water purification plant, drink food factory.More specifically, automatically can detect the bacterium in the equipment such as water receiver and pipe-line, and user can be notified or automatically take the measure such as sterilization, cleaning.In addition, can also water purification plant as device assembles on water supply line, to detect the bacterium of supplied water.
Polymer layer in described sensor is except the component parts that may be used for sensor, can also be used for make use of and have and the microorganism trap setting of the template of the three-dimensional structure of the three-dimensional arrangement complementation of microorganism, microorganism shape recognition device, microorganism follow-up mechanism, in addition, the support of the catalyst etc. that make use of porous insert can also be used for.
Embodiment
The present invention will be described by the following examples.Following embodiment is example of the present invention, does not make restriction to the present invention.
In following embodiment 1, embodiment 2, the making of polymer layer uses electrochemical gaging system (Model842B, ALS society manufactures) carry out, reference electrode employs Ag/AgCl (saturated KCl), Pt rod (diameter 1mm is employed to electrode (pair of electrodes), length 4cm, (strain) ニ ラ コ manufactures).Hereinafter, the value of what current potential was recorded is current potential relative to this reference electrode.In addition, quartz-crystal resonator (the electrode area 0.196cm being provided with gold electrode (detection electrode and second pair of electrode) on two sides is employed
2, fundamental vibration frequency 9MHz, AT cut, square, and (strain) セ イ コ ー イ ー ジ ー ア Application ド ジ ー manufactures).
As the microorganism of the detected object in embodiment 1, embodiment 2, employ Pseudomonas aeruginosa (Pseudomonasaeruginosa, Zeta potential :-33.87mV).Fig. 4 represents the electron micrograph of Pseudomonas aeruginosa.
The making > of < sensor
Embodiment 1
(the roughened operation of gold electrode)
In order to improve the tack with polypyrrole layer, the surface of gold electrode is carried out to the roughened process of the gold electrode surfaces of quartz-crystal resonator duplexer according to following step.
1. utilize plasma-etching apparatus (SEDE/meiwafosis), carry out etching in 30 seconds on gold electrode (trade(brand)name: QA-A9M-AU, (strain) セ イ コ ー イ ー ジ ー ア Application ド ジ ー manufactures) surface.
2. quartz-crystal resonator is arranged on the bottom of the room ((strain) セ イ コ ー イ ー ジ ー ア Application ド ジ ー manufactures for well type room, trade(brand)name: QA-CL4) 27 of qcm sensor 33 as shown in Figure 3.Thereafter, the solution 500 μ l of citric acid protection gold nano grain (0.0574wt%) containing 30nm is added in room 27, at room temperature places 24 hours.
3. after gold electrode being cleaned with pure water, by cetyl trimethylammonium bromide solution (0.1M) 9ml, tetrachloro gold (III) sour (salt gold (III) Suan tetra-salt compound) (0.01M) 250 μ l, NaOH (0.1M) 50 solution (gold nano grain growth media) the 500 μ l that is hybridly prepared into of μ l and xitix (0.1M) 50 μ l add in room 27, at room temperature leave standstill 24 hours.
4. remove the solution in room 27, clean gold electrode with ultrapure water.
(possessing the making of the polypyrrole layer of the template of microorganism)
On gold electrode, polypyrrole layer has been made according to following step.
5. preparation is containing 1 × 10
90.1M pyrroles's aqueous solution of cfu/ml Pseudomonas aeruginosa is as modification solution.
6. to being provided with according in the above-mentioned room 27 implementing the qcm sensor 33 of the gold electrode of roughened process, adding and modifying solution, in modification solution, insert first pair of electrode and reference electrode.
7. in modification solution, by controlled potential eletrolysis (+0.975V, 90 seconds), polypyrrole is separated out on gold electrode, make polypyrrole layer (polymerization process), after this, the polypyrrole layer by sterile water wash.In polymerization process, also carry out the monitoring of the resonant frequency of quartz-crystal resonator.To the polypyrrole layer after polymerization process, carry out surface observation by scanning electronic microscope (SEM).The thickness of polypyrrole layer is about 0.6 μm.
8. prepare EDTA solution (400 μ g/ml, pH:8.07, Tris damping fluid), in this EDTA solution, add N,O-Diacetylmuramidase dissolve, preparation lysozyme soln (20mg/ml).In addition, preparation contains the Triton solution (20wt%, pH8.03, Tris damping fluid) of nonionogenic tenside (trade(brand)name: Triton) simultaneously.
9. the lysozyme soln 250 μ l of preparation is added in room 27, at room temperature leave standstill one day, further, Triton solution is instilled 250 μ l, at room temperature leave standstill one day, bacteriolyze process (destruction operation) is carried out to the microorganism being captured to polypyrrole layer.
10. remove the solution in room 27, after using sterile water wash polypyrrole layer, use scanning electronic microscope (SEM) to carry out surface observation.
Embodiment 2
In " 8. " of above-described embodiment 1, preparation is not containing the lysozyme soln (20mg/ml) of EDTA; In " 9. ", use this lysozyme soln, in addition, make polypyrrole layer in the same manner as in Example 1.
< uses SEM to carry out the surface observation > of polypyrrole layer
Fig. 5 represents the electron micrograph on the polypyrrole layer surface after the polymerization process in embodiment 1.Fig. 5 (b) is an electron micrograph part of Fig. 5 (a) being amplified display.In Fig. 5, observe the appearance that Pseudomonas aeruginosa is caught into polypyrrole layer surface.
Fig. 6 (a), Fig. 6 (b) are after the destruction operation of embodiment 1, use the electron micrograph on the polypyrrole layer surface after sterile water wash; Fig. 6 (b) is that a part of Fig. 6 (a) amplifies the electron micrograph shown.Fig. 7 (a), Fig. 7 (b) are after the destruction operation of embodiment 2, use the electron micrograph on the polypyrrole layer surface after sterile water wash; Fig. 7 (b) is that a part of Fig. 7 (a) amplifies the electron micrograph shown.Compared with Fig. 7, in Fig. 6, there is captured Pseudomonas aeruginosa hardly, knownly in polypyrrole layer, form Pseudomonas aeruginosa template.In addition, known: (in (b), although also there is Pseudomonas aeruginosa in the part on polypyrrole layer surface, to form template at Fig. 7 (a), Fig. 7 simultaneously.
The detection > of < microorganism
(test experience)
Use the qcm sensor made as mentioned above to carry out the detection of microorganism, described qcm sensor possesses the quartz-crystal resonator defining polypyrrole layer on surface in the bottom of room.The sample solution containing microorganism is with the addition of in indoor.Then, the resonant frequency of quartz-crystal resonator has been monitored.
(result)
Fig. 8 represents the figure of the variation of resonant frequency of the quartz-crystal resonator in the sensor of embodiment 1.From the result shown in Fig. 8, in the sensor of embodiment 1, compared with the sample containing Acinetobacter calcoaceticus (Acinetobactercalcoaceticus) or blank, when with the addition of the sample solution containing Pseudomonas aeruginosa (Pseudomonasaeruginosa), resonant frequency significantly reduces.The reduction of resonant frequency means the increase of the quality on quartz-crystal resonator surface, can think, because Pseudomonas aeruginosa is captured in the template of polypyrrole layer, the quality on quartz-crystal resonator surface is increased.Therefore, the sensor of known use embodiment 1 can detect Pseudomonas aeruginosa.
Fig. 9 represents the figure of the variation of resonant frequency of the quartz-crystal resonator in the sensor of embodiment 2.From the result shown in Fig. 9, in the sensor of embodiment 2, even if add the sample solution containing Pseudomonas aeruginosa (Pseudomonasaeruginosa), do not observe the reduction of resonant frequency yet.But, from Fig. 7 (a), Fig. 7 (b), template is formed at the surface of polypyrrole layer, therefore, being interpreted as: when adopting the detection method more highly sensitive than detection method used herein, using the sensor of embodiment 2 also can detect Pseudomonas aeruginosa.
If use sensor of the present invention, such as, can rapidly and easily detect microorganism in food processing plant, therefore the reduction of defective products rate and the reduction etc. of keeping cost is not only contributed to, can also be detected by the template of the microorganism desired by formation, thus easily grasp being mixed into path and formulating counte-rplan etc. of microorganism.
Nomenclature
11 detection electrodes, 12 solution, 13 microorganisms, 14 polymer layers, 15 templates, 16 pairs of electrodes (pair of electrodes), 17 test sections, 21 controllers, 22 oscillatory circuits, 23 pairs of electrodes (second pair of electrode), 24 quartz wafers, Room 27,30 reference electrodes, 31 sample solutions, 32 quartz-crystal resonators, 33QCM sensor.
Claims (9)
1. detect the sensor of microorganism, described sensor is for test section, described test section comprises detection electrode and is arranged on the polymer layer on described detection electrode, and described polymer layer has the template with the three-dimensional structure of the three-dimensional arrangement complementation of the microorganism of detected object
The trap state that described sensor is captured to described template based on described microorganism detects described microorganism,
Described polymer layer is that the manufacture method by comprising following operation is formed:
Polymerization process, under the existence of the microorganism as detected object, by monomer polymerization, described detection electrode forms the described polymer layer of catching the state of described microorganism; And
Destroy operation, the solution of the enzyme of contact at least partially containing bacteriolyze of the microorganism being captured to described polymer layer is destroyed.
2. sensor according to claim 1, wherein, the solution used in described destruction operation is further containing sequestrant.
3. sensor according to claim 1 and 2, wherein,
Described sensor also comprises quartz-crystal resonator, described quartz-crystal resonator using the described detection electrode of described test section as in electrode,
By the change of the resonant frequency of described quartz-crystal resonator, measure the quality change of described polymer layer to detect the trap state of described microorganism.
4. according to the sensor in claim 1-3 described in any one, wherein, described monomer is selected from the group be made up of pyrroles, aniline, thiophene and their derivative.
5. sensor according to claim 4, wherein, described monomer is made up of pyrroles's or derivatives thereof.
6. according to the sensor in claim 1-5 described in any one, wherein, the forming surface of the described polymer layer of described detection electrode is asperities.
7. according to the sensor in claim 1-6 described in any one, wherein, the entirety of described microorganism or the electric charge on surface are the state of negative charge surplus.
8. one kind is detected the manufacture method of the sensor of microorganism, described sensor is for test section, described test section comprises detection electrode and is arranged on the polymer layer on detection electrode, and described polymer layer has the template with the three-dimensional structure of the three-dimensional arrangement complementation of the microorganism of detected object
Described manufacture method comprises:
Polymerization process, under the described microorganism as detected object exists, by monomer polymerization, described detection electrode forms the described polymer layer of catching the state of described microorganism; And
Destroy operation, the solution of the enzyme of contact at least partially containing bacteriolyze of the microorganism being captured to described polymer layer is destroyed.
9. a polymer layer, described polymer layer has the template with the three-dimensional structure of the three-dimensional arrangement complementation of microorganism,
Described polymer layer is that the manufacture method by comprising following operation manufactures:
Polymerization process, under the existence of described microorganism, by monomer polymerization, forms described polymer layer; And
Destroy operation, the solution of the enzyme of contact at least partially containing bacteriolyze of the microorganism being captured to described polymer layer is destroyed.
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| JP2013069739 | 2013-03-28 | ||
| JP2013-069739 | 2013-03-28 | ||
| PCT/JP2014/056143 WO2014156584A1 (en) | 2013-03-28 | 2014-03-10 | Microorganism detection sensor, method for manufacturing same, and polymer layer |
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| US (1) | US20160018391A1 (en) |
| EP (1) | EP2980204A4 (en) |
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| EP3139166A4 (en) * | 2014-05-02 | 2017-04-19 | Osaka Prefecture University Public Corporation | Polymer film for detection of cancer cells and manufacturing method thereof, and cancer cell detection device using such polymer film |
| JP6589547B2 (en) * | 2015-10-20 | 2019-10-16 | 株式会社リコー | Droplet forming device |
| CN106645348B (en) * | 2016-12-23 | 2019-03-05 | 南开大学 | A kind of preparation method of high stable microorganism electrochemical sensor |
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| US20030138845A1 (en) * | 2001-08-23 | 2003-07-24 | Changming Li | Protein and peptide sensors using electrical detection methods |
| JP2007319103A (en) * | 2006-06-02 | 2007-12-13 | Hitachi Plant Technologies Ltd | Microorganism separation system |
| JP5007905B2 (en) | 2007-08-29 | 2012-08-22 | 公立大学法人大阪府立大学 | Sensor with polymer with molecular template |
| WO2012121229A1 (en) * | 2011-03-08 | 2012-09-13 | 公立大学法人大阪府立大学 | Microorganism detection sensor and process for manufacturing same |
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2014
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| Title |
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| 涂顺明 等: "《食品杀菌新技术》", 31 January 2004 * |
| 迟玉杰 编著: "《蛋制品加工技术》", 31 January 2009 * |
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